The invention described herein was made in the course of work under a grant from the National Science Foundation.
An enzymatic activity in milk which is characterized by its activity to oxidize sulfhydryl groups of cysteine, glutathione (GSH), and milk proteins to corresponding disulfides has been demonstrated by Freidrick Kiermeier and Ernst Petz [Z. Lebensm.-Unters.-Forsch., Vol. 132, pages 342-351 (1967); and Vol. 134, pages 97-102 and 149-156 (1967)]. The reactions catalyzed by the crude preparations were suggested to be given by the equation: EQU 2RSH + 1/2 O.sub.2 .fwdarw. RSSR + H.sub.2 O.
in accordance with the general rules for systematic and trivial nomenclature, they termed the enzyme sulfhydryl oxidase.
The crude enzyme was obtained by Kiermeier and Petz from the whey fraction of skim milk. Their attempts to purify and isolate the sulfhydryl oxidase enzyme were unsuccessful.
I have now found a method for isolating and purifying sulfhydryl oxidase enzyme from milk which consistently yields preparations of greater than 3000-fold purification over skim milk. Sulfhydryl oxidase in a substantially purified form has been found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins using oxygen as an oxidant. The enzyme in a substantially purified as well as immobilized form has been found useful in treating milk to remove the cooked flavor therefrom as described in my copending U.S. application Ser. No. 621,631, filed concurrently herewith titled "PROCESS OF REMOVING THE COOKED FLAVOR FROM HEAT TREATED MILK USING IMMOBILIZED SULFHYDRYL OXIDASE ENZYME" (the entire contents of which is incorporated herein by reference). It also appears that the immobilized enzyme could be useful in the biosynthesis of disulfides in certain proteins.
Accordingly, it is the primary object of my invention to provide a method for purifying and isolating sulfhydryl oxidase enzyme from milk.
It is a further object of my invention to provide a substantially purified sulfhydryl oxidase enzyme having a specific activity which is substantially and surprisingly greater than that found in previous crude enzyme preparations.
A still yet further object of the present invention is to provide a substantially pure sulfhydryl oxidase enzyme in an immobilized form.
Still yet a further object of my invention is a means by which the activity of substantially pure sulfhydryl oxidase in immobilized form may be maintained and rejuvenated.
And still another object of the present invention is to provide a means for providing a sulfhydryl oxidase enzyme having a specific activity at least 100 times greater than that of skim milk.
These and other objects of the present invention will be more readily apparent from the description which follows.